京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22136号 / 生博第423号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 原田 浩, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

<div>Pulsed electric fields (PEFs) can induce numerous biophysical phenomena, especially perturbation of the outer and inner membranes, that may be used for applications that include nonthermal pasteurization, enhanced permeabilization of tumors to improve the transport of chemotherapeutics for cancer therapy, and enhanced membrane permeabilization of individual cells to enhance RNA and DNA delivery for gene therapy. The applied electric field and pulse duration determine the density, size, and reversibility of the created membrane pores. PEFs with durations longer than the outer membrane’s charging time will induce pore formation with the potential for application in irreversible electroporation for cancer therapy and microorganism inactivation. Shorter duration PEFs, particularly on the nanosecond timescale (nsPEFs), induce a larger density of smaller membrane pores with the potential to permeabilize intracellular membranes, such as the mitochondria, to induce programmed cell death. Thus, the PEFs can effectively kill multiple types of cells, dependent upon the cells. This thesis assesses the ability of nsPEFs to kill different cell types, specifically microorganisms with and without antibiotics as well as varying the parameters to affect populations of immortalized leukemia cells (Jurkats).</div><div>Antibiotic resistance has been an acknowledged challenge since the initial development of penicillin; however, recent discoveries by the CDC and the WHO of microorganisms resistant to last line of defense drugs combined with predictions of potential infection cases reaching 50 million a year globally and the absence new drugs in the discovery pipeline highlight the need to develop novel ways to combat and overcome these resistance mechanisms. Repurposing drugs, exploring nature for new drugs, and developing enzymes to counter the resistance mechanisms may provide potential alternatives for addressing the scarcity of antibiotics effective against gram-negative infections. One may also leverage the abundance of drugs effective against gram-positive infections by using nsPEFs to make them effective against gram-negative infections, including bacterial species with multiple natural and acquired resistance mechanisms. Numerous drug and microbial combinations for different doses and pulse treatments were tested and presented here.</div><div>Low intensity PEFs may selectively target cell populations at different stages of the cell cycle (quiescence and mitosis) to modify cancer cell population dynamics. Experimental studies of cancer cell growth when exposed to a low number of nsPEFs, while varying pulse duration, field intensity and number of pulses reveals a threshold beyond which cell recovery is not possible, but also a point of diminishing returns if cell death is the intention. A theory comprised of coupled differential equations representing the proliferating and quiescent cells showed how changing PEF parameters altered the behavior of these cell populations after treatment. These results may provide important information on the impact of PEFs with sub-threshold intensities and durations on cell population growth and potential recurrence.</div>

<p>The pancreas is a vital organ that secretes key metabolic hormones and digestive enzymes. In pancreatic ductal adenocarcinoma (PDAC), one of the leading causes of cancer-related death in the world, limited advances in diagnosis or therapies have been made over decades. Key features of PDAC progression include an elevated matrix stiffness and an increased deposition of extracellular matrices (ECM), such as hyaluronic acid (HA). Understanding how cells interact with components in the tumor microenvironment (TME) as PDAC progresses can assist in developing diagnostic tools and therapeutic treatment options. In recent years, hydrogels have proven to be an excellent platform for studying cell-cell and cell-matrix interactions. Utilizing chemically modified and naturally derived materials, hydrogel networks can be formed to encompass not only the components, but also the physicochemical properties of the dynamic TME. In this work, a dynamic hydrogel system that integrates multiple click chemistries was developed for tuning matrix physicochemical properties in a manner similar to the temporally increased matrix stiffness and depositions of HA. Subsequently, these dynamic hydrogels were used to investigate how matrix stiffening and increased HA presentation might affect survival of PDAC cells and their response to chemotherapeutics. </p>

<p>Triple Negative Breast Cancer (TNBC) is the most aggressive type of breast cancer. TNBC patients are resistant to virtually all target therapies and suffer a higher post-chemotherapy relapse with a worse overall survival compared with other types of breast cancers. Therefore, the development of an effective therapy is urgently needed. PKM2 plays a prominent role in mediating<b> </b>tumor glycolysis and PKM2 is often overexpressed in human cancers. However, whether PKM2 mediated glycolysis is necessary for cancer cell growth is questionable. Here, I have found that inhibition of PKM2 does not affect TNBC cell growth due to a metabolic switch from glycolysis to fatty acid oxidation (FAO). We show that PKM2 directly interacts with EZH2 to coordinately mediate epigenetic silencing of SLC16A9, transporter of a key player in FAO, Carnitine. Inhibition of either PKM2 or EZH2 increases levels of SLC16A9 and intracellular Carnitine to promote FAO and thereby sustains cancer cell growth. Direct inhibition of EZH2 using a clinically tested EZH2 inhibitor, GSK126, is able to elicit a previously unidentified vulnerability to a clinically tested FAO inhibitor, Etomoxir. As a result, combined GSK126-Etomoxir treatment synergistically abolishes TNBC xenograft tumor growth in vivo. Together, this study uncovers PKM2-EZH2 mediated metabolic reprogramming that leads to a new drug combination therapy by dual targeting of EZH2 and FAO for effective treatment of TNBC.<b> </b></p> <p> </p> <p>Furthermore, Dendritic Cell (DC) vaccination has shown promise in treating cancer patients. However, the <i>in vitro</i> generation of a fully functional DC remains a big challenge in this field. EZH2 inhibition has shown to be able to create an immunologically ‘hot’ tumors. Nonetheless, the role of EZH2 in regulation of DC function is still unclear. I found that the expression levels of EZH2 and its functional maker, H3K27Me3, are enhanced following maturation from immature DC (iDC) into two functional DCs, α-type 1-polarized-DC (αDC) and gold standard DC (sDC). Moreover, inhibition of EZH2 by GSK126 treatment elicits a dependency of sDC on FAO. These results suggest that EZH2 plays a role in maturation of DC through metabolic reprogramming, which may also provide new DC based immunotherapy of TNBC. </p>

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lucie Čermáková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: The effect of acalabrutinib and ibrutinib on the efficacy of daunorubicin in cancer cells Leukemia presents malignant diseases of hematopoiesis, which essence is the malignant transformation of a hematopoietic stem cell at various levels of maturation and increased proliferative activity. Chemotherapy is the gold standard in the treatment of leukemia. One of the many treatments is the use of anthracycline chemotherapeutics, especially daunorubicin (DAU). Anthracyclines are widely used in clinical practice but have high cardiotoxic effects that limit their dosage. One of the main causes of side effects is the reduction of an anthracycline chemotherapeutic to the appropriate toxic metabolite, which accumulates in the heart. Carbonyl, reducing enzymes from the superfamily aldo-ketoreductase (AKR), and short-chain dehydrogenase/reductase (SDR) are involved in this reduction. At the same time, carbonyl reducing enzymes, has been shown to be involved in the mechanisms that cause tumor cells to be resistant to anthracyclines, thereby reducing the inhibition of the growth of these cells. In the diploma thesis we found that...

<p>Epigenetic mechanisms, including DNA methylation, play an important role in regulation of stem cell fate and tumorigenesis. The Ten-Eleven-Translocation 2 (TET2) is a core enzyme for DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydromethylcytosine (5hmC). It has been shown that TET2 is the main regulator of hematopoietic stem cell homeostasis and loss of TET2 is highly associated with hematopoietic malignancies. Our previous work has also shown that loss of TET2 expression is linked to promotion of an epithelial-mesenchymal-transition phenotype and expansion of a breast cancer stem cell-like population with skewed asymmetric cell division in vitro; however, the in vivo role that TET2 plays in regulation of mammary stem cell (MaSC) fate and development of mammary pathology has yet to be determined. Here, using our newly established mammary-specific Tet2-knockout mouse model, the data reveals for the first time that TET2 plays a pivotal role in mammary gland development via directing MaSC to luminal lineage commitment in vivo. Furthermore, we find that TET2 coordinates with FOXP1 to target and demethylate FOXA1, GATA3, and ESR1, key transcription factors that orchestrate mammary luminal lineage specification and endocrine response and are often silenced by DNA methylation in aggressive human breast cancers. Finally, loss of TET2 expression leads to promotion of mammary tumor development with defective luminal cell differentiation and tamoxifen resistance in a PyMT;Tet2 deletion breast cancer mouse model. As a result, this study provides a previously unidentified role for TET2 in governing luminal lineage specification and endocrine response that underlies resistance to anti-estrogen treatments.</p>

<p>Breast cancer is the most frequently diagnosed malignancy and the second most lethal cancer in women. Metastasis in breast cancer is invariably responsible for patient death and is comprised of many steps, of which proliferation in vital organs is responsible for morbidity and mortality due to vital organ failure. Patients with the metastatic disease are limited to chemotherapy, which non-specifically targets proliferating cells. Despite it being initially effective, chemotherapy is associated with high toxicity and many patients develop resistance. Thus, there is an urgent need to characterize the biology of metastatic breast cancer to develop targeted therapies for the late-stage disease.</p> <p>EGFR is a member of the ErbB family of receptor tyrosine kinases, which have particular relevance in breast tumorigenesis. Clinical studies show that high expression levels of EGFR in the primary mammary tumors correlate with poor prognosis and decreased survival of breast cancer patients due to metastasis. Patient data is supported by experimental and pre-clinical studies, which describe various signaling pathways that mediate the oncogenic effects of EGFR, such as the MAPK, STAT3, and PI3K pathways. Despite these well-documented roles of EGFR, clinical trials evaluating EGFR inhibitors (EGFRi) in metastatic breast cancer have been unanimously unsuccessful in improving patient prognosis, and the mechanisms that contribute to this intrinsic resistance are unknown.</p> <p>To characterize the signaling events that govern EGFR behavior in metastatic breast cancer resistant to EGFRi, we utilized multiple pre-clinical breast cancer progression series and patient-derived cells that display the intrinsic resistance phenomenon. In these models, EGFR functions as a pro-apoptotic molecule whose ligand-mediated activation results in growth inhibition and/or apoptosis of metastatic breast cancer cells. Here we show that in the later stages of metastasis, increased nuclear translocation of EGFR leads to increased physical access to STAT1 and STAT3 molecules residing in the nucleus. Indeed, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1/3 phosphorylation. Additionally, specific inhibition of nuclear EGFR function using the EGFR kinase inhibitor gefitinib linked to a nuclear localization signal (NLS-gefitinib) prevents EGF-induced STAT1/3 phosphorylation. Altogether, these findings implicate nuclear localization of EGFR in downstream STAT1/3 signaling in metastatic breast cancer.</p> <p>Subsequently, we examined the involvement of nuclearly-activated STAT1/3 signaling in the apoptotic function of EGFR. NLS-gefitinib treatment or genetic/pharmacologic inhibition of STAT1/3 efficiently blocks EGF-induced apoptosis in metastatic breast cancer cells resistant to EGFRi. These findings were utilized therapeutically by activating EGFR with EGF treatment while simultaneously blocking the downstream proliferative MAPK:ERK1/2 pathway using the MEK1/2 inhibitor trametinib. EGF + trametinib combination preserved STAT1 signaling while effectively blocking the MAPK pathway, thus potentiating EGF-mediated apoptosis in metastatic breast cancer cells. Importantly, combined administration of trametinib and EGF resulted in STAT1-mediated apoptosis of primary mammary tumor cells, which respond to EGF in a proliferative fashion. These data provide a novel approach of targeting metastatic breast cancer by biasing EGFR signaling towards nuclear activation of STAT1/3 signaling resulting in apoptosis.</p> Our studies herein also examined the role of STAT3 in primary mammary tumor cells overexpressing EGFR. Depletion of STAT3 expression normalized the transformed phenotype of these cells <i>in vitro</i> and resulted in smaller mammary tumors <i>in vivo</i>. These results implicate STAT3 in EGFR-driven breast tumorigenesis localized to the mammary tissues. Further, systemic dissemination of breast cancer is associated with activation of the JAK1/2:STAT3 signaling axis. Despite the involvement of STAT3 in EGFR-mediated oncogenesis in the primary tumor setting, targeting JAK1/2:STAT signaling with the JAK1/2 inhibitor ruxolitinib proved ineffective in inhibiting the growth and invasion of metastatic cells derived from these primary tumors. These results are in agreement with the role of STAT1/3 in driving the pro-apoptotic function of EGFR in metastatic breast cancer cells. Altogether, these investigations provide a plausible explanation for the inability of JAK1/2 inhibitors to effectively target metastatic breast cancer in clinical and experimental investigations. Further, these findings indicate that the development of therapeutics or molecular tools that efficiently activate STAT1/3 signaling in metastatic breast cancer may represent an important concept for eradicating tumors resistant to targeted therapies.

Thrombotic events are known to be associated with various cancers and recent research has implicated parts of the coagulation systemin promoting cancer progression. In particular, thrombin has been studied for its mitogenic effects in 2D cultures as well as in cancer progression in vivo in animal models however, conflicting results exist. Studies of proliferation in response to thrombin stimulation, of pancreatic cancer cells or pancreatic cancer-associated fibroblasts (CAFs) in vitro, that utilize a3D culture platform are significantly limited. In this study, PDAC cancer cells and cancer-associated fibroblast (CAF) cells were exposed to thrombin using a microfluidic device that mimics in vivo conditions. The cells used herein were cultured in a microfluid device, suspended inside of a 3D collagen matrix, and exposed to daily stimulation of 1 U/mL of thrombin in serum-free media for one hour. The findings of this study are that there is no statistically significant effect, promotive or inhibitory, on the proliferation of the cells used in this study, these results were unexpected. At the end of this paper, a review of potential reasons as to why no significant effect was seen on the cells is presented.

<p>Breast cancer is the predominant cancer diagnosed among women, and the second most deadly cancer. The vast majority of cancer-related deaths is caused by the metastatic spread of cancer from the primary tumor to a distant site in the body. Therefore, new strategies which minimize breast cancer metastasis are imperative to improve patient survival. Cancer cells which acquire anchorage independence, or the ability to survive without extracellular matrix attachment, and metabolic flexibility have increased potential to metastasize. In the present studies, the ability to survive detachment and subsequent metabolic changes were determined in human Harvey-<i>ras</i> transformed MCF10A-<i>ras</i> breast cancer cells. Detachment resulted in reduced viability in a time-dependent manner with the lowest cell viability observed at forty hours. In addition, decreased cell viability was observed in both glutamine and glucose depleted detached conditions, suggesting a dependence on both nutrients for detached survival. Compared to attached cells, detached cells had reduced total pool sizes of pyruvate, lactate, α-ketoglutarate, fumarate, malate, alanine, serine, and glutamate, suggesting the metabolic stress which occurs under detached conditions. However, intracellular citrate and aspartate pools were unchanged, demonstrating a preference to maintain these pools in detached conditions. Compared to attached cells, detached cells had suppressed glutamine metabolism, as determined by decreased glutamine flux into the TCA cycle and reduced mRNA abundance of glutamine metabolizing enzymes. Further, detached glucose anaplerosis through pyruvate dehydrogenase activity was decreased, while pyruvate carboxylase (PC) expression and activity were increased. A switch in metabolism was observed away from glutamine anaplerosis to a preferential utilization of PC activity to replenish the TCA cycle, determined by reduced PC mRNA abundance in detached cells treated with a cell-permeable analog of α-ketoglutarate, the downstream metabolite of glutamine which enters the TCA cycle. These results suggest that detached cells elevate PC to increase flux of carbons into the TCA cycle when glutamine metabolism is reduced. </p> <p>Vitamin D is recognized for its role in preventing breast cancer progression, and recent studies suggest that regulation of energy metabolism may contribute to its anticancer effects. Vitamin D primarily acts on target tissue through its most active metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)₂D). The present work investigated 1,25(OH)₂D’s effects on viability of detached cells through regulation of energy metabolism. Treatment of MCF10A-<i>ras</i> cells with 1,25(OH)₂D resulted in decreased viability of detached cells. While 1,25(OH)₂D treatment did not affect many of the glucose metabolism outcomes measured, including intracellular pyruvate and lactate pool sizes, glucose flux to pyruvate and lactate, and mRNA abundance of enzymes involved in glucose metabolism, 1,25(OH)₂D treatment reduced detached PC expression and glucose flux through PC. A reduction in glutamine metabolism was observed with 1,25(OH)₂D treatment, although no 1,25(OH)₂D target genes were identified. Further, PC depletion by shRNA decreased cell viability in detached conditions with no additional effect with 1,25(OH)₂D treatment. Moreover, PC overexpression resulted in increased detached cell viability and inhibited 1,25(OH)₂D’s negative effects on viability. These results suggest that 1,25(OH)₂D reduces detached cell viability through regulation of PC. Collectively this work identifies a key metabolic adaptation where detached cells increase PC expression and activity to compensate for reduced glutamine metabolism and that 1,25(OH)₂D may be utilized to reverse this effect and decrease detached cell viability. These results contribute to an increased understanding of metastatic processes and the regulation of these processes by vitamin D, which may be effective in preventing metastasis and improve breast cancer patient survival.</p>

A simple method is reported to synthesize an anticancer drug, NCX 4040, conveniently in mild conditions using silicon chemistry. A starting material, 4-hydroxybenzyl alcohol, was silylated selectively first to give t-butyldimethylsilyl 4-hydroxybenzyl ether, which was then converted to NCX 4040 by esterification, desilylation, hydrochlorination and nitration.